Benzylisoquinoline Alkaloid (BIA) Precursor Producing Microbes, and Methods of Making and Using the Same

ABSTRACT

Host cells that are engineered to produce benzylisoquinoline alkaloid (BIAs) precursors, such as norcodaurine (NC) and norlaudanosoline (NL), are provided. The host cells may have one or more engineered modifications selected from: a feedback inhibition alleviating mutation in a enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a BIA of interest or a precursor thereof using the host cells and compositions, e.g., kits, systems etc., that find use in methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/899,496filed on Nov. 4, 2013; the disclosure of which application is hereinincorporated by reference.

GOVERNMENT RIGHTS

This invention is made with Government support under grant No. 1066100awarded by the National Science Foundation. The Government has certainrights in this invention.

INTRODUCTION

Benzylisoquinoline alkaloids (BIAs) are a large group of secondarymetabolites from plants and other organisms. These molecules havetherapeutic functions in the human body, ranging from the establishedanalgesic and antitussive properties of morphine and codeine, to novelactivities against cancer and infection observed for molecules such asberberine and sanguinarine. Supply of all these BIA molecules so thatthey are available to researchers and physicians is of interest. Thenumber of synthetic reactions and requirements for selectivestereochemistry means that chemical synthesis of BIAs is low yieldingand not a viable means for large-scale production. Instead, for thewidely used drugs codeine and morphine, the opium poppy (Papaversomniferum) has been bred and developed as a production crop.Intermediates in morphine biosynthesis that find use as drugs and drugprecursors do not accumulate because the plant metabolism is evolved tomaximize pathway flux to the final opioids. Even for end productmetabolites like morphine, accumulation occurs only within specializedcells in the buds and vascular tissue and requires harsh chemicalprocessing of harvested plant material during the extraction process,which may yield less than 2% morphine by dry weight. As such, methodsfor preparing BIAs are of interest.

SUMMARY

Host cells that are engineered to produce benzylisoquinoline alkaloid(BIA) precursors, such as norcoclaurine (NC) and norlaudanosoline (NL),are provided. The host cells may have one or more modifications selectedfrom: a feedback inhibition alleviating mutation in an enzyme gene; atranscriptional modulation modification of a biosynthetic enzyme gene;an inactivating mutation in an enzyme; and a heterologous codingsequence. Also provided are methods of producing a BIA of interest or aprecursor thereof using the host cells and compositions, e.g., kits,systems etc., that find use in methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 illustrates the biosynthetic pathway from glucose to tyrosine andother BIA precursor molecules.

FIG. 2 illustrates the effect of ZWF1 knockout and TKL1 over-expressionon the pentose phosphate pathway (PPP). A: native PPP flux, B: ModifiedPPP flux.

FIG. 3 illustrates the synthesis of NC (A) and NL (B) from precursormolecules.

FIG. 4 illustrates the effect of four genetic modifications on NCproduction with varying fed tyrosine.

FIG. 5 shows NC production from strains with combinations of geneticmodifications.

FIG. 6 shows the levels of NL production in aldehyde oxidoreductase(ALD) alcohol dehydrogenase (ADH) gene knockout strains FIG. 7illustrates the activity of a L-DOPA decarboxylase (DODC) enzyme invivo. Yeast strains transformed with DNA to express Papaver somniferumtyrosine/DOPA decarboxylase can convert L-DOPA to dopamine in vivo.

FIG. 8 shows the production of norcoclaurine (NC) in yeast strains fed100 mM dopamine and varying concentrations of tyrosine.

FIG. 9 shows NC production in multiple engineered yeast strains fed 100mM dopamine and no tyrosine.

FIG. 10 shows NC production from dopamine or from L-DOPA in anengineered yeast strain (CSY980) with the additional integration of theL-DOPA decarboxylase PpDODC.

FIG. 11 illustrates a biosynthetic scheme including tyrosinehydroxylation using mammalian tyrosine hydroxylases (TyrHs) with theco-substrate tetrahydrobiopterin (BH4).

FIG. 12 shows that tyrosine hydroxylases expressed from yeast cellsconvert tyrosine to L-DOPA: (A) LC-MS chromatogram confirms conversionof tyrosine to L-DOPA in the presence of co-substrate, BH4; and (B)L-DOPA ion fragmentation in lysate samples.

FIG. 13 shows the co-expression of tyrosine hydroxylase with a BH4biosynthetic enzyme provides for conversion of tyrosine to L-DOPA.

FIG. 14 illustrates the synthesis of the BIA precursor moleculescoclaurine and N-methylcoclaurine from NC.

FIG. 15 shows LC-MS analysis (A: ion counts) of the production ofNC-derived BIA precursor molecules including N-methylcoclaurine (B: m/zfragmentation pattern) from L-DOPA in the liquid culture of engineeredyeast strains.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY. Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims is drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the terms “determining.” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length.e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein the term “isolated,” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

As used herein, the term “encoded by” refers to a nucleic acid sequencewhich codes for a polypeptide sequence, wherein the polypeptide sequenceor a portion thereof contains an amino acid sequence of 3 or more aminoacids, such as 5 or more, 8 or more, 10 or more, 15 or more or 20 ormore amino acids from a polypeptide encoded by the nucleic acidsequence. Also encompassed by the term are polypeptide sequences thatare immunologically identifiable with a polypeptide encoded by thesequence.

A “vector” is capable of transferring gene sequences to target cells. Asused herein, the terms, “vector construct,” “expression vector.” and“gene transfer vector.” are used interchangeably to mean any nucleicacid construct capable of directing the expression of a gene of interestand which can transfer gene sequences to target cells, which isaccomplished by genomic integration of all or a portion of the vector,or transient or inheritable maintenance of the vector as anextrachromosomal element. Thus, the term includes cloning, andexpression vehicles, as well as integrating vectors.

An “expression cassette” includes any nucleic acid construct capable ofdirecting the expression of a gene/coding sequence of interest, which isoperably linked to a promoter of the expression cassette. Such cassettesis constructed into a “vector.” “vector construct,” “expression vector,”or “gene transfer vector,” in order to transfer the expression cassetteinto target cells. Thus, the term includes cloning and expressionvehicles, as well as viral vectors.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 10 or more, such as 100 or more, 1000 or more, 10,000 or more,100.000 or more, 100 or more, 10⁷ or more, 10⁸ or more or 10⁹ or moremembers.

Numeric ranges are inclusive of the numbers defining the range.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Host cells that are engineered to produce benzylisoquinoline alkaloid(BIAs) precursors, such as norcoclaurine (NC) and norlaudanosoline (NL),are provided. The host cells may have one or more engineeredmodifications selected from: a feedback inhibition alleviating mutationin a enzyme gene; a transcriptional modulation modification of abiosynthetic enzyme gene; an inactivating mutation in an enzyme; and aheterologous coding sequence. Also provided are methods of producing aBIA of interest or a precursor thereof using the host cells andcompositions, e.g., kits, systems etc., that find use in methods of theinvention.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context dearly dictates otherwise, between the upper and lower limitof that range and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method is carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, benzylisoquinoline alkaloidprecursors of interest are described first in greater detail, followedby host cells for producing the same. Next, methods of interest in whichthe host cells find use are reviewed. Kits that may be used inpracticing methods of the invention are also described.

Benzylisoquinoline Alkaloid (BIA) Precursors

As summarized above, host cells which produce benzylisoquinolinealkaloid precursors (BIA precursors) are provided. The BIA precursor maybe any intermediate or precursor compound in a synthetic pathway (e.g.,as described herein) that leads to the production of a BIA of interest(e.g., as described herein). In some cases, the BIA precursor has astructure that may be characterized as a BIA or a derivative thereof. Incertain cases, the BIA precursor has a structure that may becharacterized as a fragment of a BIA. In some cases, the BIA precursoris an early BIA. As used herein, by “early BIA” is meant an earlyintermediate in the synthesis of a BIA of interest in a cell, where theearly BIA is produced by a host cell from a host cell feedstock orsimple starting compound. In some cases, the early BIA is a BIAintermediate that is produced by the subject host cell solely from ahost cell feedstock (e.g., a carbon and nutrient source) without theneed for addition of a starting compound to the cells. The term earlyBIA may refer to a precursor of a BIA end product of interest whether ornot the early BIA can be itself be characterized as a benzylisoquinolinealkaloid.

In some cases, the BIA precursor is an early BIA, such as apre-reticuline benzylisoquinoline alkaloid. As such, host cells whichproduce pre-reticuline benzylisoquinoline alkaloids (pre-reticulineBIAs) are provided. Reticuline is a major branch point intermediate ofinterest in the synthesis of downstream BIAs via cell engineeringefforts to produce end products such as opioid products. The subjecthost cells may produce BIA precursors from simple and inexpensivestarting materials that may find use in the production of reticuline anddownstream BIA end products.

As used herein, the terms “pre-reticuline benzylisoquinoline alkaloid”,“pre-reticuline BIA” and “pre-reticuline BIA precursor” are usedinterchangeably and refer to a biosynthetic precursor of reticulinewhether or not the structure of the reticuline precursor itself ischaracterized as a benzylisoquinoline alkaloid. The term pre-reticulineBIA is meant to include biosynthetic precursors, intermediates andmetabolites thereof, of any convenient member of a host cellbiosynthetic pathway that may lead to reticuline. In some cases, thepre-reticuline BIA includes a benzylisoquinoline alkaloid fragment, suchas a benzyl fragment, a quinoline fragment or a precursor or derivativethereof. In certain instances, the pre-reticuline BIA has a structurethat can be characterized as a benzylisoquinoline alkaloid or aderivative thereof.

BIA precursors of interest include, but are not limited to,norcoclaurine (NC) and norlaudanosoline (NL), as well as NC and NLprecursors, such as tyrosine, 4-hydroxyphenylacetaldehyde (4-HPA),4-hydroxyphenylpyruvic acid (4-HPPA), L-3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine. Insome embodiments, the one or more BIA precursors are3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In certaininstances, the one or more BIA precursors are4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. FIGS. 3A and 3Billustrate the synthesis of NC and NL, respectively from precursormolecules via a Pictet-Spengler condensation reaction, where thereaction may occur spontaneously or may by catalyzed by any convenientenzymes.

Synthetic pathways to a BIA precursor may be generated in the hostcells, and may start with any convenient starting compound(s) ormaterials. FIG. 1 illustrates a synthetic pathway of interest to BIAprecursors starting from glucose. The starting material may benon-naturally occurring or the starting material may be naturallyoccurring in the host cell. Any convenient compounds and materials maybe used as the starting material, based upon the synthetic pathwaypresent in the host cell. The source of the starting material may befrom the host cell itself, e.g., tyrosine, or the starting material maybe added or supplemented to the host cell from an outside source. Assuch, in some cases, the starting compound refers to a compound in asynthetic pathway of the cell that is added to the host cell from anoutside source that is not part of a growth feedstock or cell growthmedia. Starting compounds of interest include, but are not limited to,dopamine, 4-HPA, 4-HPPA, as well as any of the compounds shown inFIG. 1. For example, if the host cells are growing in liquid culture,the cell media may be supplemented with the starting material, which istransported into the cells and converted into the desired products bythe cell. Starting materials of interest include, but are not limitedto, inexpensive feedstocks and simple precursor molecules. In somecases, the host cell utilizes a feedstock including a simple carbonsource as the starting material, which the host cell utilizes to producecompounds of the synthetic pathway of the cell. The host cell growthfeedstock may include one or more components, such as a carbon sourcesuch as cellulose, starch, free sugars and a nitrogen source, such asammonium salts or inexpensive amino acids. In some cases, a growthfeedstock that finds use as a starting material may be derived from asustainable source, such as biomass grown on marginal land, includingswitchgrass and algae, or biomass waste products from other industrialor farming activities.

Host Cells

As summarized above, one aspect of the invention is a host cell thatproduces one or more BIA precursors. Any convenient cells may beutilized in the subject host cells and methods. In some cases, the hostcells are non-plant cells. In some instances, the host cells may becharacterized as microbial cells. In certain cases, the host cells areinsect cells, mammalian cells, bacterial cells or yeast cells. Anyconvenient type of host cell may be utilized in producing the subjectBIA-producing cells, see, e.g., US2008/0176754, and US2014/0273109 thedisclosures of which are incorporated by reference in their entirety.Host cells of interest include, but are not limited to, bacterial cells,such as Bacillus subtilis, Escherichia coli, Streptomyces and Salmonellatyphimuium cells, insect cells such as Drosophila melanogaster S2 andSpodoptera frugiperda Sf9 cells and yeast cells such as S. cerevisiaecells, Schizosaccharomyces pombe cells and a Pichia pastoris cells. Insome embodiments, the host cells are yeast cells or E. coli cells. Insome cases, the host cell is a yeast cell. In some instances the hostcell is from a strain of yeast engineered to produce a BIA precursor ofinterest. Any of the host cells described in US200810176754, andUS2014/0273109 by Smolke et al. may be adapted for use in the subjectcells and methods. In certain embodiments, the yeast cells can be of thespecies Saccharomyces cerevisiae (S. cerevisiae). In certainembodiments, the yeast cells can be of the species Schizosaccharomycespombe. In certain embodiments, the yeast cells can be of the speciesPichia pastoris. Yeast is of interest as a host cell because cytochromeP450 proteins, which are involved in some biosynthetic pathways ofinterest, are able to fold properly into the endoplasmic reticulummembrane so that their activity is maintained. Yeast strains of interestthat find use in the invention include, but are not limited to, CEN.PK(Genotype: MATa/α ura3-52/ura3-52 trp1-28 trp-289 leu2-3_112/leu2-3_112his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2), S288C, W303, D273-108,X2180, A364A, Σ1278B, AB972, SK1 and FL100. In certain cases, the yeaststrain is any of S288C (MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1),BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1;leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa/MATα; his3Δ1/his3Δ1;leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0; ura3Δ0/ura3Δ0), and WAT11 orW(R), derivatives of the W303-B strain (MATa; ade2-1; his3-11, -15;leu2-3, -112; ura3-1; canR; cyr+) which express the Arabidopsis thalianaNADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1,respectively. In another embodiment, the yeast cell is W303alpha (MATa;his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype ofadditional yeast strains of interest can be found at EUROSCARF(web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).

The host cells may be engineered to include one or more modifications(such as two or more, three or more, four or more, five or more, or evenmore modifications) that provide for the production of BIA precursors ofinterest. In some cases, by modification is meant a geneticmodification, such as a mutation, addition or deletion of a gene orfragment thereof, or transcription regulation of a gene or fragmentthereof. In some cases, the one or more (such as two or more, three ormore or four or more) modifications is selected from: a feedbackinhibition alleviating mutation in a biosynthetic enzyme gene native tothe cell; a transcriptional modulation modification of a biosyntheticenzyme gene native to the cell; an inactivating mutation in an enzymenative to the cell; and a heterologous coding sequence that encodes anenzyme. A cell that includes one or more modifications may be referredto as a modified cell.

A modified cell may overproduce one or more BIA precursor molecules. Byoverproduce is meant that the cell has an improved or increasedproduction of a BIA precursor molecule of interest relative to a controlcell (e.g., an unmodified cell). By improved or increased production ismeant both the production of some amount of the BIA precursor ofinterest where the control has no BIA precursor production, as well asan increase of about 10% or more, such as about 20% or more, about 30%or more, about 40% or more, about 50% or more, about 60% or more, about80% or more, about 100% or more, such as 2-fold or more, such as 5-foldor more, including 10-fold or more in situations where the control hassome BIA precursor production.

In some cases, the host cell is capable of producing an increased amountof norcocdaurine relative to a control host cell that lacks the one ormore modifications (e.g., as described herein). In certain instances,the increased amount of norcodaurine is about 10% or more relative tothe control host cell, such as about 20% or more, about 30% or more,about 40% or more, about 50% or more, about 60% or more, about 80% ormore, about 100% or more, 2-fold or more, 5-fold or more, or even10-fold or more relative to the control host cell.

In some cases, the host cell is capable of producing an increased amountof norlaudonosoline relative to a control host cell that lacks the oneor more modifications (e.g., as described herein). In certain instances,the increased amount of norlaudonosoline is about 10% or more relativeto the control host cell, such as about 20% or more, about 30% or more,about 40% or more, about 50% or more, about 60% or more, about 80% ormore, about 100% or more, 2-fold or more, 5-fold or more, or even10-fold or more relative to the control host cell.

In some embodiments, the host cell is capable of producing a 10% or moreyield of norcoclaurine from a starting compound such as tyrosine, suchas 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, or even 90% or more yield of norcodaurine from astarting compound.

In some embodiments, the host cell is capable of producing a 10% or moreyield of norlaudonosoline from a starting compound such as tyrosine,such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, or even 90% or more yield of norlaudonosolinefrom a starting compound.

In some embodiments, the host cell overproduces one or more BIAprecursor molecule selected from the group consisting of tyrosine,4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dinydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine.

Any convenient combinations of the one or more modifications may beincluded in the subject host cells. In some cases, two or more (such astwo or more, three or more, or four or more) different types ofmodifications are included. In certain instances, two or more (such asthree or more, four or more, five or more, or even more) distinctmodifications of the same type of modification are included in thesubject cells.

In some embodiments of the host cell, when the cell includes one or moreheterologous coding sequences that encode one or more enzymes, itincludes at least one additional modification selected from the groupconsisting of: a feedback inhibition alleviating mutations in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and an inactivating mutation in an enzyme native to the cell. Incertain embodiments of the host cell, when the cell includes one or morefeedback inhibition alleviating mutations in one or more biosyntheticenzyme genes native to the cell, it includes a least one additionalmodification selected from the group consisting of: a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; an inactivating mutation in an enzyme native to the cell; and aheterologous coding sequence that encode an enzyme. In some embodimentsof the host cell, when the cell includes one or more transcriptionalmodulation modifications of one or more biosynthetic enzyme genes nativeto the cell, it includes at least one additional modification selectedfrom the group consisting of: a feedback inhibition alleviating mutationin a biosynthetic enzyme gene native to the cell; an inactivatingmutation in an enzyme native to the cell; and a heterologous codingsequence that encodes an enzyme. In certain instances of the host cell,when the cell includes one or more inactivating mutations in one or moreenzymes native to the cell, it includes at least one additionalmodification selected from the group consisting of: a feedbackinhibition alleviating mutation in a biosynthetic enzyme gene native tothe cell; a transcriptional modulation modification of a biosyntheticenzyme gene native to the cell; and a heterologous coding sequence thatencodes an enzyme.

In certain embodiments of the host cell, the cell includes one or morefeedback inhibition alleviating mutations in one or more biosyntheticenzyme genes native to the cell; and one or more transcriptionalmodulation modifications of one or more biosynthetic enzyme gene nativeto the cell. In certain embodiments of the host cell, the cell includesone or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the cell; and one or moreinactivating mutations in an enzyme native to the cell. In certainembodiments of the host cell, the cell includes one or more feedbackinhibition alleviating mutations in one or more biosynthetic enzymegenes native to the cell; and one or more heterologous coding sequences.In some embodiments, the host cell includes one or more modifications(e.g., as described herein) that include one or more of the genes ofinterest described in Table 1.

Feedback Inhibition Alleviating Mutations

In some instances, the host cells are cells that include one or morefeedback inhibition alleviating mutations (such as two or more, three ormore, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some cases, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). As used herein, the term “feedback inhibitionalleviating mutation” refers to a mutation that alleviates a feedbackinhibition control mechanism of a host cell. Feedback inhibition is acontrol mechanism of the cell in which an enzyme in the syntheticpathway of a regulated compound is inhibited when that compound hasaccumulated to a certain level, thereby balancing the amount of thecompound in the cell. In some instances, the one or more feedbackinhibition alleviating mutations is in an enzyme described in asynthetic pathway of FIG. 1 or FIG. 2. A mutation that alleviatesfeedback inhibition reduces the inhibition of a regulated enzyme in thecell of interest relative to a control cell and provides for anincreased level of the regulated compound or a downstream biosyntheticproduct thereof. In some cases, by alleviating inhibition of theregulated enzyme is meant that the IC₅₀ of inhibition is increased by2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold ormore, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold ormore, or even more. By increased level is meant a level that is 110% ormore of that of the regulated compound in a control cell or a downstreamproduct thereof, such as 120% or more, 130% or more, 140% or more, 150%or more, 160% or more, 170% or more, 180% or more, 190% or more or 200%or more, such as at least 3-fold or more, at least 5-fold or more, atleast 10-fold or more or even more of the regulated compound in the hostcell or a downstream product thereof.

A variety of feedback inhibition control mechanisms and biosyntheticenzymes native to the host cell that are directed to regulation oflevels of BIA precursors may be targeted for alleviation in the hostcell. The host cell may include one or more feedback inhibitionalleviating mutations in one or more biosynthetic enzyme genes native tothe cell. The mutation may be located in any convenient biosyntheticenzyme genes native to the host cell where the biosynthetic enzyme issubject to regulatory control. In some embodiments, the one or morebiosynthetic enzyme genes encode one or more enzymes selected from a3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and achorismate mutase. In some embodiments, the one or more biosyntheticenzyme genes encode a 3-deoxy-d-arabinose-heptulosonate-7-phosphate(DAHP) synthase. In some instances, the one or more biosynthetic enzymegenes encode a chorismate mutase. In certain instances, the one or morefeedback inhibition alleviating mutations are present in a biosyntheticenzyme gene selected from ARO4 and ARO7. In certain instances, the oneor more feedback inhibition alleviating mutations are present in abiosynthetic enzyme gene that is ARO4. In certain instances, the one ormore feedback inhibition alleviating mutations are present in abiosynthetic enzyme gene that is ARO7. In some embodiments, the hostcell includes one or more feedback inhibition alleviating mutations inone or more biosynthetic enzyme genes such as one of those genesdescribed in Table 1.

Any convenient numbers and types of mutations may be utilized toalleviate a feedback inhibition control mechanism. As used herein, theterm “mutation” refers to a deletion, insertion, or substitution of anamino acid(s) residue or nucleotide(s) residue relative to a referencesequence or motif. The mutation may be incorporated as a directedmutation to the native gene at the original locus. In some cases, themutation may be incorporated as an additional copy of the geneintroduced as a genetic integration at a separate locus, or as anadditional copy on an episomal vector such as a 2μ or centromericplasmid. In certain instances, the feedback inhibited copy of the enzymeis under the native cell transcriptional regulation. In some instances,feedback inhibited copy of the enzyme is introduced with engineeredconstitutive or dynamic regulation of protein expression by placing itunder the control of a synthetic promoter.

In certain embodiments, the one or more feedback inhibition alleviatingmutations are present in the ARO4 gene. ARO4 mutations of interestinclude, but are not limited to, substitution of the lysine residue atposition 229 with a leucine, a substitution of the glutamine residue atposition 166 with a lysine residue, or a mutation as described byHartmann M, at al. ((2003) Proc Natl Acad Sci USA 100(3):862-887) orFukuda et al. ((1992) J Ferment Bioeng 74(2):117-119). In someinstances, mutations for conferring feedback inhibition are selectedfrom a mutagenized library of enzyme mutants. Examples of suchselections include rescue of growth of o-fluoro-D,L-phenylalanine orgrowth of aro3 mutant yeast strains in media with excess tyrosine asdescribed by Fukuda et al. ((1990) Breeding of Brewing Yeast Producing aLarge Amount of Beta-Phenylethyl Alcohol and Beta-Phenylethyl Acetate.Agr Biol Chem Tokyo 54(1):269-271).

ARO7 mutations of interest include, but are not limited to, substitutionof the threonine residue at position 226 with an isoleucine, asdescribed by Schmidheini at al. ((1989), J Bacteriol 171(3):1245-1253)and additional mutations conferring feedback inhibition selected from amutagenized library of microbial chorismate mutase mutants. Examples ofsuch selections include assays for 5-methyltryptophan sensitivity orincreased production of melanin pigments in strains expressingheterologous tyrosinase enzymes (1.9) in the absence of externally fedtyrosine.

In certain embodiments, the host cells of the present invention mayinclude 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, or even 15 or more feedback inhibitionalleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the host cell.

Transcriptional Modulation Modifications

The host cells may include one or more transcriptional modulationmodifications (such as two or more, three or more, four or more, five ormore, or even more modifications) of one or more biosynthetic enzymegenes of the cell. In some cases, the one or more biosynthetic enzymegenes are native to the cell. Any convenient biosynthetic enzyme genesof the cell may be targeted for transcription modulation. Bytranscription modulation is meant that the expression of a gene ofinterest in a modified cell is modulated, e.g., increased or decreased,enhanced or repressed, relative to a control cell (e.g., an unmodifiedcell). In some cases, transcriptional modulation of the gene of interestincludes increasing or enhancing expression. By increasing or enhancingexpression is meant that the expression level of the gene of interest isincreased by 2-fold or more, such as by 5-fold or more and sometimes by25-, 50-, or 100-fold or more and in certain embodiments 300-fold ormore or higher, as compared to a control, i.e., expression in the samecell not modified (e.g., by using any convenient gene expression assay).Alternatively, in cases where expression of the gene of interest in acell is so low that it is undetectable, the expression level of the geneof interest is considered to be increased if expression is increased toa level that is easily detectable. In certain instances, transcriptionalmodulation of the gene of interest includes decreasing or repressingexpression. By decreasing or repressing expression is meant that theexpression level of the gene of interest is decreased by 2-fold or more,such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or moreand in certain embodiments 300-fold or more or higher, as compared to acontrol. In some cases, expression is decreased to a level that isundetectable. Modifications of host cell processes of interest that maybe adapted for use in the subject host cells are described in U.S.Publication No. 20140273109 (Ser. No. 14/211,611) by Smolke et al., thedisclosure of which is herein incorporated by reference in its entirety.

Any convenient biosynthetic enzyme genes may be transcriptionallymodulated, and include but are not limited to, those biosyntheticenzymes described in FIG. 1, such as ARO3, ARO4. ARO1, ARO7, TYR1. TYR,TyrH, DODC, MAO, ARO10, ARO9 and TKL. In some instances, the one or morebiosynthetic enzyme genes is selected from ARO10, ARO9 and TKL. In somecases, the one or more biosynthetic enzyme genes is ARO10. In certaininstances, the one or more biosynthetic enzyme genes is ARO9. In someembodiments, the one or more biosynthetic enzyme genes is TKL. In someembodiments, the host cell includes one or more transcriptionalmodulation modifications to one or more genes such as one of those genesdescribed in Table 1. In some embodiments, the host cell includes one ormore transcriptional modulation modifications to one or more genes suchas one of those genes described in a synthetic pathway of one of FIGS. 1and 2.

In some embodiments, the transcriptional modulation modificationincludes substitution of a strong promoter for a native promoter of theone or more biosynthetic enzyme genes. The promoters driving expressionof the genes of interest can be constitutive promoters or induciblepromoters, provided that the promoters can be active in the host cells.The genes of interest may be expressed from their native promoters, ornon-native promoters may be used. Although not a requirement, suchpromoters should be medium to high strength in the host in which theyare used. Promoters may be regulated or constitutive. In someembodiments, promoters that are not glucose repressed, or repressed onlymildly by the presence of glucose in the culture medium, are used. Thereare numerous suitable promoters, examples of which include promoters ofglycolytic genes such as the promoter of the B. subtilis tsr gene(encoding fructose biphosphate aldolase) or GAPDH promoter from yeast S.cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase) (BitterG. A., Meth. Enzymol. 152:673 684 (1987)). Other strong promoters ofinterest include, but are not limited to, the ADHI promoter of baker'syeast (Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)), thephosphate-starvation induced promoters such as the PHO5 promoter ofyeast (Hinnen, A., et al, in Yeast Genetic Engineering. Barr, P. J., etal. eds, Butterworths (1989), the alkaline phosphatase promoter from B.licheniformis (Lee. J. W. K., et al., J. Gen. Microbiol, 137:1127 1133(1991)), GPD1 and TEF1. Yeast promoters of interest include, but are notlimited to, inducible promoters such as Gal1-10. Gal1, GalL, GalS,repressible promoter Met25, tetO, and constitutive promoters such asglyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcoholdehydrogenase promoter (ADH), translation-elongation factor-1-alphapromoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter,etc. In some instances, the strong promoter is GPD1. In certaininstances, the strong promoter is TEF1. Autonomously replicating yeastexpression vectors containing promoters inducible by hormones such asglucocorticoids, steroids, and thyroid hormones are also known andinclude, but are not limited to, the glucorticoid responsive element(GRE) and thyroid hormone responsive element (TRE), see e.g., thosepromoters described in U.S. Pat. No. 7,045,290. Vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. Additionally any promoter/enhancercombination (as per the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression of genes of interest. It is understood thatany convenient promoters specific to the host cell may be selected.e.g., E. coli. In some cases, promoter selection can be used to optimizetranscription, and hence, enzyme levels to maximize production whileminimizing energy resources.

Inactivating Mutations

The host cells may include one or more inactivating mutations to anenzyme of the cell (such as two or more, three or more, four or more,five or more, or even more). The inclusion of one or more inactivatingmutations may modify the flux of a synthetic pathway of a host cell toincrease the levels of a BIA precursor of interest or a desirable enzymeor precursor leading to the same. In some cases, the one or moreinactivating mutations are to an enzyme native to the cell FIG. 2illustrates a native pentose phosphate pathway (PPP) flux and modifiedPPP flux where that involves inactivation of ZWF1 enzyme. As usedherein, by “inactivating mutation” is meant one or more mutations to agene or regulatory DNA sequence of the cell, where the mutation(s)inactivates a biological activity of the protein expressed by that geneof interest. In some cases, the gene is native to the cell. In someinstances, the gene encodes an enzyme that is inactivated and is part ofor connected to the synthetic pathway of a BIA precursor produced by thehost cell. In some instances, an inactivating mutation is located in aregulatory DNA sequence that controls a gene of interest. In certaincases, the inactivating mutation is to a promoter of a gene. Anyconvenient mutations (e.g., as described herein) may be utilized toinactivate a gene or regulatory DNA sequence of interest. By“inactivated” or “inactivates” is meant that a biological activity ofthe protein expressed by the mutated gene is reduced by 10% or more,such as by 20% or more, 30% or more, 40% or more, 50% or more, 80% ormore, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more or99% or more, relative to a control protein expressed by a non-mutatedcontrol gene. In some cases, the protein is an enzyme and theinactivating mutation reduces the activity of the enzyme.

In some embodiments, the cell includes an inactivating mutation in anenzyme native to the cell. Any convenient enzymes may be targeted forinactivation. Enzymes of interest include, but are not limited to thoseenzymes, described in FIGS. 1 and 2 whose action in the syntheticpathway of the host cell tends to reduce the levels of a BIA precursorof interest. In some cases, the enzyme has glucose-6-phosphatedehydrogenase activity. In certain embodiments, the enzyme that includesan inactivating mutation is ZWF1 (see e.g., FIG. 2). In some cases, theenzyme has alcohol dehydrogenase activity. In some embodiments, theenzyme that includes an inactivating mutation is selected from ADH2,ADH3, ADH4, ADH5, ADH6. ADH7 and SFA1. In certain embodiments, theenzyme that includes an inactivating mutation(s) is ADH2. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH3. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ADH4. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ADH5. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ADH6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. Incertain embodiments, the enzyme that includes an inactivating mutationis selected from ALD2, ALD3, ALD4, ALD5 and ALD6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD2. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ALD3. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ALD4. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ALD5. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD6. In some embodiments, the host cell includes one or moreinactivating mutations to one or more genes described in Table 1.

Heterologous Coding Sequences

In some instances, the host cells are cells that harbor one or moreheterologous coding sequences (such as two or more, three or more, fouror more, five or more, or even more) which encode activity(ies) thatenable the host cells to produce desired BIA precursor(s), e.g., asdescribed herein. As used herein, the term “heterologous codingsequence” is used to indicate any polynucleotide that codes for, orultimately codes for, a peptide or protein or its equivalent amino acidsequence, e.g., an enzyme, that is not normally present in the hostorganism and can be expressed in the host cell under proper conditions.As such. “heterologous coding sequences” includes multiple copies ofcoding sequences that are normally present in the host cell, such thatthe cell is expressing additional copies of a coding sequence that arenot normally present in the cells. The heterologous coding sequences canbe RNA or any type thereof, e.g., mRNA, DNA or any type thereof. e.g.,cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, butare not limited to, full-length transcription units that include suchfeatures as the coding sequence, introns, promoter regions, 3′-UTRs andenhancer regions.

In some embodiments, the host cell includes norcoclaurine (NC) synthaseactivity. Any convenient NC synthase enzymes find use in the subjecthost cells. NC synthase enzymes of interest include, but are not limitedto, enzymes such as EC 4.2.1.78, as described in Table 1. In certainembodiments, the host cell includes a heterologous coding sequence foran NC synthase or an active fragment thereof. In some instances, thehost cell includes one or more heterologous coding sequences for one ormore enzymes or active fragments thereof that convert tyrosine toL-DOPA. In certain cases, the one or more enzymes is selected frombacterial tyrosinases, eukaryotic tyrosinases (e.g., EC 1.14.18.1) andtyrosine hydroxylases (e.g., EC 1.14.16.2.) In some instances, the hostcell includes one or more heterologous coding sequences for one or moreenzymes or active fragments thereof that convert L-DOPA to dopamine(e.g., EC 4.1.1.28).

In certain embodiments, the cell includes one or more heterologouscoding sequences for one or more enzymes or active fragments thereofthat convert dopamine to 3,4-DHPA. In certain cases, the one or moreenzymes is a monoamine oxidase (MAO) (e.g., EC 1.4.3.4). The one or moreheterologous coding sequences may be derived from any convenient species(e.g., as described herein). In some cases, the one or more heterologouscoding sequences may be derived from a species described in Table 1. Insome cases, the one or more heterologous coding sequences are present ina gene or enzyme selected from those described in Table 1.

In some instances, the one or more heterologous coding sequences includea MAO coding sequence integrated at a genomic locus encoding nativeARO10. In certain instances, the one or more heterologous codingsequences include a MAO coding sequence operably linked to an induciblepromoter. In some embodiments, the inducible promoter is part of aninducible system including a DNA binding protein targeted to a promoterregulating the ARO10 gene. In some embodiments, the host cell includesone or heterologous coding sequences for one or more enzymes or activefragments thereof described in the genes of Table 1.

As used herein, the term “heterologous coding sequences” also includesthe coding portion of the peptide or enzyme, i.e., the cDNA or mRNAsequence, of the peptide or enzyme, as well as the coding portion of thefull-length transcriptional unit, i.e., the gene including introns andexons, as well as “codon optimized” sequences, truncated sequences orother forms of altered sequences that code for the enzyme or code forits equivalent amino acid sequence, provided that the equivalent aminoacid sequence produces a functional protein. Such equivalent amino acidsequences can have a deletion of one or more amino acids, with thedeletion being N-terminal, C-terminal or internal. Truncated forms areenvisioned as long as they have the catalytic capability indicatedherein. Fusions of two or more enzymes are also envisioned to facilitatethe transfer of metabolites in the pathway, provided that catalyticactivities are maintained.

Operable fragments, mutants or truncated forms may be identified bymodeling and/or screening. This is made possible by deletion of, forexample, N-terminal, C-terminal or internal regions of the protein in astep-wise fashion, followed by analysis of the resulting derivative withregard to its activity for the desired reaction compared to the originalsequence. If the derivative in question operates in this capacity, it isconsidered to constitute an equivalent derivative of the enzyme proper.

Aspects of the present invention also relate to heterologous codingsequences that code for amino acid sequences that are equivalent to thenative amino acid sequences for the various enzymes. An amino acidsequence that is “equivalent” is defined as an amino acid sequence thatis not identical to the specific amino acid sequence, but rathercontains at least some amino acid changes (deletions, substitutions,inversions, insertions, etc.) that do not essentially affect thebiological activity of the protein as compared to a similar activity ofthe specific amino acid sequence, when used for a desired purpose. Thebiological activity refers to, in the example of a decarboxylase, itscatalytic activity. Equivalent sequences are also meant to include thosewhich have been engineered and/or evolved to have properties differentfrom the original amino acid sequence. Mutable properties of interestinclude catalytic activity, substrate specificity, selectivity,stability, solubility, localization, etc. In certain embodiments, an“equivalent” amino acid sequence contains at least 80%-99% identity atthe amino acid level to the specific amino acid sequence, in some casesat least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and morein certain cases, at least 95%, 986%, 97%, 98% and 99% identity, at theamino acid level. In some cases, the amino acid sequence may beidentical but the DNA sequence is altered such as to optimize codonusage for the host organism, for example.

The host cells may also be modified to possess one or more geneticalterations to accommodate the heterologous coding sequences.Alterations of the native host genome include, but are not limited to,modifying the genome to reduce or ablate expression of a specificprotein that may interfere with the desired pathway. The presence ofsuch native proteins may rapidly convert one of the intermediates orfinal products of the pathway into a metabolite or other compound thatis not usable in the desired pathway. Thus, if the activity of thenative enzyme were reduced or altogether absent, the producedintermediates would be more readily available for incorporation into thedesired product.

In some instances, where ablation of expression of a protein may be ofinterest, is in proteins involved in the pleiotropic drug response,including, but not limited to, ATP-binding cassette (ABC) transporters,multidrug resistance (MDR) pumps and associated transcription factors.These proteins are involved in the export of BIA molecules into theculture medium, thus deletion controls the export of the compounds intothe media, making them more available for incorporation into the desiredproduct. In some embodiments, host cell gene deletions of interestinclude genes associated with the unfolded protein response andendoplasmic reticulum (ER) proliferation. Such gene deletions may leadto improved BIA production. The expression of cytochrome P450s mayinduce the unfolded protein response and may cause the ER toproliferate. Deletion of genes associated with these stress responsesmay control or reduce overall burden on the host cell and improvepathway performance. Genetic alterations may also include modifying thepromoters of endogenous genes to increase expression and/or introducingadditional copies of endogenous genes. Examples of this include theconstruction/use of strains which overexpress the endogenous yeastNADPH-P450 reductase CPR1 to increase activity of heterologous P450enzymes. In addition, endogenous enzymes such as ARO8, 9, and 10, whichare directly involved in the synthesis of intermediate metabolites, mayalso be overexpressed.

Heterologous coding sequences of interest include but are not limited tosequences that encode enzymes, either wild-type or equivalent sequences,that are normally responsible for the production of BIAs and precursorsin plants. In some cases, the enzymes for which the heterologoussequences code can be any of the enzymes in the BIA pathway, and can befrom any convenient source. The choice and number of enzymes encoded bythe heterologous coding sequences for the particular synthetic pathwaymay be selected based upon the desired product. In certain embodiments,the host cells of the present invention may include 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 8 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,or even 15 or more heterologous coding sequences, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 heterologous coding sequences.

In some cases, peptide sequences encoded by the heterologous codingsequences are as reported in GENBANK. Enzymes of interest include, butare not limited to, those enzymes described herein and those shown inTable 1. The host cells may include any combination of the listedenzymes, from any source. Unless otherwise indicated, accession numbersin Table 1 refer to GenBank. Some accession numbers refer to theSaccharomyces genome database (SGD), which is available on theworld-wide web at www.yeastgenome.org.

In some embodiments, the host cell (e.g., a yeast strain) is engineeredfor selective production of a BIA of interest by localizing one or moreenzymes to a compartment in the cell. In some cases, an enzyme may belocated in the host cell such that the compound produced by this enzymespontaneously rearranges, or is converted by another enzyme to adesirable metabolite before reaching a localized enzyme that may convertthe compound into an undesirable metabolite. The spatial distancebetween two enzymes may be selected to prevent one of the enzymes fromacting directly on a compound to make an undesirable metabolite, andrestrict production of undesirable end products (e.g., an undesirableopioid by-product). In certain embodiments, any of the enzymes describedherein, either singularly or together with a second enzyme, may belocalized to any convenient compartment in the host cell, including butnot limited to, an organelle, endoplasmic reticulum, golgi, vacuole,nucleus, plasma membrane or the periplasm. In some embodiments, the hostcell includes one or more of the enzymes that include a localizationtag. Any convenient tags may be utilized. In some cases, thelocalization tag is a peptidic sequence that is attached at theN-terminal and or C-terminal of the enzyme.

Any convenient methods may be utilized for attaching a tag to theenzyme. In some cases, the localization tag is derived from anendogenous yeast protein. Such tags may provide route to a variety ofyeast organelles: the endoplasmic reticulum (ER), mitochondria (MT),plasma membrane (PM), and vacuole (V). In certain embodiments, the tagis an ER routing tag (e.g., ER1). In certain embodiments, the tag is avacuole tag (e.g., V1). In certain embodiments, the tag is a plasmamembrane tag (e.g., P1). In certain instances, the tag includes or isderived from, a transmembrane domain from within the tail-anchored classof proteins. In some embodiments, the localization tag locates theenzyme on the outside of an organelle. In certain embodiments, thelocalization tag locates the enzyme on the inside of an organelle.

In some instances, the expression of each type of enzyme is increasedthrough additional gene copies (i.e., multiple copies), which increasesintermediate accumulation and/or BIA precursor production. Embodimentsof the present invention include increased BIA precursor production in ahost cell through simultaneous expression of multiple species variantsof a single or multiple enzymes. In some cases, additional gene copiesof a single or multiple enzymes are included in the host cell. Anyconvenient methods may be utilized including multiple copies of aheterologous coding sequence for an enzyme in the host cell.

In some embodiments, the host cell includes multiple copies of aheterologous coding sequence for an enzyme, such as 2 or more, 3 ormore, 4 or more, 5 or more, or even 10 or more copies. In certainembodiments, the host cell include multiple copies of heterologouscoding sequences for one or more enzymes, such as multiple copies of twoor more, three or more, four or more, etc. In some cases, the multiplecopies of the heterologous coding sequence for an enzyme are derivedfrom two or more different source organisms as compared to the hostcell. For example, the host cell may include multiple copies of oneheterologous coding sequence, where each of the copies is derived from adifferent source organism. As such, each copy may include somevariations in explicit sequences based on inter-species differences ofthe enzyme of interest that is encoded by the heterologous codingsequence.

In some embodiments of the host cell, the heterologous coding sequenceis from a source organism selected from the group consisting of P.somniferum, T. flavum and C. japonica. In some instances, the sourceorganism is P. somniferum, E. caldomica, C. japonica, T. flavum,Berberis stolonifer, T. flavum subsp. glaucum, Coptis chinensis,Thalictrum spp, Coptis spp, Papaver spp, Berberis wilsonae, A. mexicana,or Berberis spp. In certain instances, the heterologous coding sequenceis from a source organism selected from P. somniferum, T. flavum and C.japonica. In some embodiments, the host cell includes a heterologouscoding sequence from one or more of the source organisms described inTable 1.

The engineered host cell medium may be sampled and monitored for theproduction of BIA precursors of interest. The BIA precursors may beobserved and measured using any convenient methods. Methods of interestinclude, but are not limited to, LC-MS methods (e.g., as describedherein) where a sample of interest is analyzed by comparison with aknown amount of a standard compound. Identity may be confirmed, e.g.: bym/z and MS/MS fragmentation patterns, and quantitation or measurement ofthe compound may be achieved via LC trace peaks of know retention timeand/or EIC MS peak analysis by reference to corresponding LC-MS analysisof a known amount of a standard of the compound.

Methods

As summarized above, aspects of the invention include methods ofpreparing a benzylisoquinoline alkaloid (BIA) of interest. As such,aspects of the invention include culturing a host cell under conditionsin which the one or more host cell modifications (e.g., as describedherein) are functionally expressed such that the cell converts startingcompounds of interest into product BIAs of interest or precursorsthereof (e.g., pre-reticuline BIAs). Also provided are methods thatinclude culturing a host cell under conditions suitable for proteinproduction such that one or more heterologous coding sequences arefunctionally expressed and convert starting compounds of interest intoproduct BIAs of interest. In some instances, the method is a method ofpreparing a benzylisoquinoline alkaloid (BIA), include culturing a hostcell (e.g., as described herein); adding a starting compound to the cellculture; and recovering the BIA from the cell culture. In someembodiments of the method, the starting compound. BIA product and hostcell are described by one of the entries of Table 1.

Any convenient methods of culturing host cells may be employed forproducing the BIA precursors and downstream BIAs of interest. Theparticular protocol that is employed may vary, e.g., depending on hostcell, the heterologous coding sequences, the desired BIA precursors,etc. The cells may be present in any convenient environment, such as anenvironment in which the cells are capable of expressing one or morefunctional heterologous enzymes. In vitro, as used herein, simply meansoutside of a living cell, regardless of the location of the cell. Asused herein, the term in vivo indicates inside a living cell, regardlessof the location of the cell. In some embodiments, the cells are culturedunder conditions that are conducive to enzyme expression and withappropriate substrates available to allow production of BIA precursorsin vivo. In some embodiments, the functional enzymes are extracted fromthe host for production of BIAs under in vitro conditions. In someinstances, the host cells are placed back into a multicellular hostorganism. The host cells are in any phase of growth, including, but notlimited to, stationary phase and log-growth phase, etc. In addition, thecultures themselves may be continuous cultures or they may be batchcultures.

Any convenient cell culture conditions for a particular cell type may beutilized. In certain embodiments, the host cells that includes one ormore modifications is cultured under standard or readily optimizedconditions, with standard cell culture media and supplements. As oneexample, standard growth media when selective pressure for plasmidmaintenance is not required may contain 20 g/L yeast extract, 10 g/Lpeptone, and 20 g/L dextrose (YPD). Host cells containing plasmids isgrown in synthetic complete (SC) media containing 1.7 g/L yeast nitrogenbase, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with theappropriate amino acids required for growth and selection. Alternativecarbon sources which may be useful for inducible enzyme expressioninclude, but are not limited to, sucrose, raffinose, and galactose.Cells is grown at any convenient temperature (e.g., 30° C.) with shakingat any convenient rate (e.g., 200 rpm) in a vessel, e.g., in test tubesor flasks in volumes ranging from 1-1000 mL, or larger, in thelaboratory. Culture volumes can also be scaled up for growth in largerfermentation vessels, for example, as part of an industrial process.

Any convenient codon optimization techniques for optimizing theexpression of heterologous polynucleotides in host cells may be adaptedfor use in the subject host cells and methods, see e.g., Gustafsson, C.et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated byreference in its entirety.

The subject method may also include adding a starting compound to thecell culture. Any convenient methods of addition may be adapted for usein the subject methods. The cell culture may be supplemented with asufficient amount of the starting materials of interest (e.g., asdescribed herein), e.g., a mM to μM amount such as between about 1-5 mMof a starting compound. It is understood that the amount of startingmaterial added, the timing and rate of addition, the form of materialadded, etc., may vary according to a variety of factors. The startingmaterial may be added neat or pre-dissolved in a suitable solvent (e.g.,cell culture media, water or an organic solvent). The starting materialmay be added in concentrated form (e.g., 10× over desired concentration)to minimize dilution of the cell culture medium upon addition. Thestarting material may be added in one or more batches, or by continuousaddition over an extended period of time (e.g., hours or days).

The subject methods may also include recovering the BIA precursor ordownstream BIA of interest from the cell culture. Any convenient methodsof separation and isolation (e.g., chromatography methods orprecipitation methods) may be adapted for use in the subject methods torecover the BIA of interest or precursor thereof from the cell culture.Filtration methods may be used to separate soluble from insolublefractions of the cell culture. In some cases, liquid chromatographymethods (e.g., reverse phase HPLC, size exclusion, normal phasechromatography) are used to separate the BIA or precursor from othersoluble components of the cell culture. In some cases, extractionmethods (e.g., liquid extraction, pH based purification, etc.) are usedto separate the BIA precursor or BIA from other components of the cellculture.

Also included are methods of engineering host cells for the purpose ofproducing BIAs of interest or precursors thereof. Inserting DNA intohost cells may be achieved using any convenient methods. The methods areused to insert the heterologous coding sequences into the host cellssuch that the host cells functionally express the enzymes and convertstarting compounds of interest into product BIAs of interest.

Any convenient promoters may be utilized in the subject host cells andmethods. The promoters driving expression of the heterologous codingsequences may be constitutive promoters or inducible promoters, providedthat the promoters are active in the host cells. The heterologous codingsequences may be expressed from their native promoters, or non-nativepromoters may be used. Such promoters may be low to high strength in thehost in which they are used. Promoters may be regulated or constitutive.In certain embodiments, promoters that are not glucose repressed, orrepressed only mildly by the presence of glucose in the culture medium,are used. Promoters of interest include but are not limited to,promoters of glycolytic genes such as the promoter of the B. subtilistsr gene (encoding the promoter region of the fructose bisphosphatealdolase gene) or the promoter from yeast S. cerevisiae gene coding forglyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1promoter of baker's yeast, the phosphate-starvation induced promoterssuch as the PHO5 promoter of yeast, the alkaline phosphatase promoterfrom B. licheniformis, yeast inducible promoters such as Gal1-10, Gal1,GalL, GalS, repressible promoter Met25, tetO, and constitutive promoterssuch as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcoholdehydrogenase promoter (ADH), translation-elongation factor-1-α promoter(TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, etc.Autonomously replicating yeast expression vectors containing promotersinducible by hormones such as glucocorticoids, steroids, and thyroidhormones may also be used and include, but are not limited to, theglucorticoid responsive element (GRE) and thyroid hormone responsiveelement (TRE). These and other examples are described U.S. Pat. No.7,045,290, which is incorporated by reference, including the referencescited therein. Additional vectors containing constitutive or induciblepromoters such as a factor, alcohol oxidase, and PGH may be used.Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression ofgenes. Any convenient appropriate promoters may be selected for the hostcell, e.g., E. coli. One can also use promoter selection to optimizetranscript, and hence, enzyme levels to maximize production whileminimizing energy resources.

Any convenient vectors may be utilized in the subject host cells andmethods. Vectors of interest include vectors for use in yeast and othercells. The types of yeast vectors can be broken up into 4 generalcategories: integrative vectors (YIp), autonomously replicating highcopy-number vectors (YEp or 2μ plasmids), autonomously replicating lowcopy-number vectors (YCp or centromeric plasmids) and vectors forcloning large fragments (YACs). Vector DNA is introduced intoprokaryotic or eukaryotic cells via any convenient transformation ortransfection techniques.

Utility

The host cells and methods of the invention, e.g., as described above,find use in a variety of applications. Applications of interest include,but are not limited to: research applications and therapeuticapplications. Methods of the invention find use in a variety ofdifferent applications including any convenient application where theproduction of BIAs is of interest.

The subject host cells and methods find use in a variety of therapeuticapplications. Therapeutic applications of interest include thoseapplications in which the preparation of pharmaceutical products thatinclude BIAs is of interest. The host cells described herein producebenzylisoquinoline alkaloid precursors (BIA precursors). Reticuline is amajor branch point intermediate of interest in the synthesis of BIAsincluding engineering efforts to produce end products such as opioidproducts. The subject host cells may be utilized to produce BIAprecursors from simple and inexpensive starting materials that may finduse in the production of reticuline and BIA end products. As such, thesubject host cells find use in the supply of therapeutically active BIAsor precursors thereof.

In some instances, the host cells and methods find use in the productionof commercial scale amounts of BIAs or precursors thereof where chemicalsynthesis of these compounds is low yielding and not a viable means forlarge-scale production. In certain cases, the host cells and methods areutilized in a fermentation facility that would include bioreactors(fermenters) of e.g., 5,000-200,000 liter capacity allowing for rapidproduction of BIAs of interest or precursors thereof for therapeuticproducts. Such applications may include the industrial-scale productionof BIAs of interest from fermentable carbon sources such as cellulose,starch, and free sugars.

The subject host cells and methods find use in a variety of researchapplications. The subject host cells and methods may be used to analyzethe effects of a variety of enzymes on the biosynthetic pathways of avariety of BIAs of interest or precursors thereof. In addition, the hostcells may be engineered to produce BIAs or precursors thereof that finduse in testing for bioactivity of interest in as yet unproventherapeutic functions. In some cases, the engineering of host cells toinclude a variety of heterologous coding sequences that encode for avariety of enzymes elucidates the high yielding biosynthetic pathwaystowards BIAs of interest, or precursors thereof. In certain cases,research applications include the production of precursors fortherapeutic molecules of interest that can then be further chemicallymodified or derivatized to desired products or for screening forincreased therapeutic activities of interest. In some instances, hostcell strains are used to screen for enzyme activities that are ofinterest in such pathways, which may lead to enzyme discovery viaconversion of BIA metabolites produced in these strains.

The subject host cells and methods may be used as a production platformfor plant specialized metabolites. The subject host cells and methodsmay be used as a platform for drug library development as well as plantenzyme discovery. For example, the subject host cells and methods mayfind use in the development of natural product based drug libraries bytaking yeast strains producing interesting scaffold molecules, such asprotopine, and further functionalizing the compound structure throughcombinatorial biosynthesis or by chemical means. By producing druglibraries in this way, any potential drug hits are already associatedwith a production host that is amenable to large-scale culture andproduction. As another example, these subject host cells and methods mayfind use in plant enzyme discovery. The subject host cells provide aclean background of defined metabolites to express plant EST librariesto identify new enzyme activities. The subject host cells and methodsprovide expression methods and culture conditions for the functionalexpression and increased activity of plant enzymes in yeast.

Kits and Systems

Aspects of the invention further include kits and systems, where thekits and systems may include one or more components employed in methodsof the invention, e.g., host cells, starting compounds, heterologouscoding sequences, vectors, culture medium, etc., as described herein. Insome embodiments, the subject kit includes a host cell (e.g., asdescribed herein), and one or more components selected from thefollowing: starting compounds, a heterologous coding sequence and/or avector including the same, vectors, growth feedstock, componentssuitable for use in expression systems (e.g., cells, cloning vectors,multiple cloning sites (MCS), bi-directional promoters, an internalribosome entry site (IRES), etc.) and a culture medium.

Any of the components described herein may be provided in the kits,e.g., host cells including one or more modifications, startingcompounds, culture medium, etc. A variety of components suitable for usein making and using heterologous coding sequences, cloning vectors andexpression systems may find use in the subject kits. Kits may alsoinclude tubes, buffers, etc., and instructions for use. The variousreagent components of the kits may be present in separate containers, orsome or all of them may be pre-combined into a reagent mixture in asingle container, as desired.

Also provided are systems for producing a BIA of interest, where thesystems may include engineered host cells including one or moremodifications (e.g., as described herein), starting compounds, culturemedium, a fermenter and fermentation equipment, e.g., an apparatussuitable for maintaining growth conditions for the host cells, samplingand monitoring equipment and components, and the like. A variety ofcomponents suitable for use in large scale fermentation of yeast cellsmay find use in the subject systems.

In some cases, the system includes components for the large scalefermentation of engineered host cells, and the monitoring andpurification of BIA compounds produced by the fermented host cells. Incertain embodiments, one or starting compounds (e.g., as describedherein) are added to the system, under conditions by which theengineered host cells in the fermenter produce one or more desired BIAproducts or precursors thereof. In some instances, the host cellsproduce a BIA precursor of interest (e.g., as described herein). Incertain cases, the BIA products of interest are opioid products, such ascodeine, neopine, morphine, neomorphine, hydrocodone, oxycodone,hydromorphone, dihydrocodeine, 14-hydroxycodeine, or dihydromorphine.

In some cases, the system includes means for monitoring and or analyzingone or more BIA compounds or precursors thereof produced by the subjecthost cells. For example, a LC-MS analysis system as described herein, achromatography system, or any convenient system where the sample may beanalyzed and compared to a standard, e.g., as described herein. Thefermentation medium may be monitored at any convenient times before andduring fermentation by sampling and analysis. When the conversion ofstarting compounds to BIA products or precursors of interest iscomplete, the fermentation may be halted and purification of the BIAproducts may be done. As such, in some cases, the subject systemincludes a purification component suitable for purifying the BIAproducts or precursors of interest from the host cell medium into whichit is produced. The purification component may include any convenientmeans that may be used to purify the BIA products or precursors offermentation, including but not limited to, silica chromatography,reverse-phase chromatography, ion exchange chromatography. HICchromatography, size exclusion chromatography, liquid extraction and pHextraction methods. In some cases, the subject system provides for theproduction and isolation of BIA fermentation products of interestfollowing the input of one or more starting compounds to the system.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXPERIMENTAL Example I

A series of specific genetic modifications provide a biosyntheticprocess in Saccharomyces cerevisiae for the production of BIAs fromsimple, inexpensive feedstocks or precursor molecules. Methods forconstructing novel strains capable of producing the early BIA moleculesnorcodaurine (NC) and norlaudanosoline (NL) from non-BIA precursors orsimple feedstocks are described. NC has never been reported as a productof microbial synthesis and is the natural precursor to all known BIAmolecules. Methods for manipulating the regulation of yeast biosyntheticpathways and for optimizing the production of aromatic amino acids andrelated BIA precursors are also described.

A. tyrosine and Related BIA Precursor Overproducing Yeast Strains

Strains of S. cerevisiae are developed with improved flux through thearomatic amino acid biosynthesis pathway for the purposes of increasingintracellular concentrations of BIA precursor molecules includingtyrosine, 4-hydroxyphenylacetaldehyde (4-HPA),L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde(3,4-DHPA), and dopamine. These strains combine genetic modifications tothe yeast strain for the purpose of increasing carbon flux from centralmetabolism towards aromatic amino acid synthesis in general, towardstyrosine in particular, and include the introduction of key heterologousenzymes for the production of BIA precursor molecules not naturallyproduced by yeast. Genetic modifications are employed including theintroduction of feedback inhibition alleviating mutations to genesencoding native biosynthetic enzymes, tuning of transcriptionalregulation of native biosynthetic enzymes, deletion of genes encodingenzymes that divert precursor molecules away from the intended pathway,and introduction of heterologous enzymes for the conversion of naturallyendogenous molecules into non-native BIA precursor molecules.

Specific Description:

1.1) The biosynthetic pathway in the engineered strain incorporatesfeedback inhibition alleviating mutations (1.1.1) to the native yeastgene ARO4, which encodes a 3-deoxy-d-arabinose-heptulosonate-7-phosphate(DAHP) synthase, alone or in combination. This mutation (ARO4^(FBR)) isincorporated as a directed mutation to the native gene at the originallocus, as an additional copy introduced as a genetic integration at aseparate locus, or as an additional copy on an episomal vector such as a2μ or centromeric plasmid. FBR refers to feedback resistant mutants andmutations. The feedback inhibited copy of the DAHP synthase enzyme isunder the native yeast transcriptional regulation or is introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter.

1.1.1) ARO4^(FBR) mutations may include, for example, a substitution ofthe lysine residue at position 229 with a leucine (see e.g., Hartmann M,et al. (2003) Evolution of feedback-inhibited beta/alpha barrelisoenzymes by gene duplication and a single mutation. Proc Natl Acad SciUSA 100(3):862-867), a substitution of the glutamine residue at position166 with a lysine residue (see e.g., Fukuda K et al. (1992)Feedback-Insensitive Mutation of3-Deoxy-D-Arabino-Hepturosonate-7-Phosphate Synthase Caused by a SingleNucleotide Substitution of Aro4 Structural Gene inSaccharomyces-Cerevisiae. J Ferment Bioeng 74(2): 117-119), or anadditional mutation conferring feedback inhibition selected from amutagenized library of microbial DHAP synthase mutants. Examples of suchselections include rescue of growth on o-fluoro-D,L-phenylalanine (seee.g., Fukuda et al. (1990) Breeding of Brewing Yeast Producing a LargeAmount of Beta-Phenylethyl Alcohol and Beta-Phenylethyl Acetate. AgrBiol Chem Tokyo 54(1):269-271) or growth of aro3 mutant yeast strains onmedia with excess tyrosine.

1.2) The biosynthetic pathway in the engineered strain incorporates afeedback inhibition alleviating mutation (1.2.1) to the native yeastgene ARO7, which encodes the enzyme chorismate mutase. This mutation(ARO7^(FBR)) is incorporated as a directed mutation to the native geneat the original locus, as an additional copy introduced as a geneticintegration at a separate locus, or as an additional copy on an episomalvector such as a 2μ or centromeric plasmid. The feedback inhibited copyof the chorismate mutase enzyme is under the native yeasttranscriptional regulation or is introduced with engineered constitutiveor dynamic regulation of protein expression by placing it under thecontrol of a synthetic promoter.

1.2.1) ARO7^(FBR) mutant alleles may include, for example, asubstitution of the threonine residue at position 226 with an isoleucine(see e.g., Schmidheini et al. (1989) A Single Point Mutation Results ina Constitutively Activated and Feedback-Resistant Chorismate Mutase ofSaccharomyces-Cerevisiae. J Bacteriol 171(3):1245-1253) or an additionalmutation conferring feedback inhibition selected from a mutagenizedlibrary of microbial chorismate mutase mutants. Examples of suchselections include assays for 5-methyltryptophan sensitivity orincreased production of melanin pigments in strains expressingheterologous tyrosinase enzymes (1.9) in the absence of externally fedtyrosine.

1.3) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1, TEF1, etc) forthe overexpression of the native yeast gene ARO10, which encodes anenzyme with hydroxyphenylpyruvate decarboxylase activity. This geneticmodification is incorporated as a directed swapping of the nativepromoter DNA sequence at the original locus, as an additional copy ofthe gene under new transcriptional regulation introduced as a geneticintegration at a separate locus, or as an additional copy on an episomalvector such as a 2p or centromeric plasmid.

1.4) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1, TEF1, etc) forthe overexpression of the native yeast gene ARO9, which encodes anenzyme with hydroxyphenylpyruvate/glutamic acid transaminase activity.This genetic modification is incorporated as a directed swapping of thenative promoter DNA sequence at the original locus, as an additionalcopy of the gene under new transcriptional regulation introduced as agenetic integration at a separate locus, or as an additional copy on anepisomal vector such as a 2μ or centromeric plasmid.

1.5) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1. TEF1, etc) forthe overexpression of the native yeast gene TKL, which encodes an enzymewith transketolase activity. This genetic modification is incorporatedas a directed swapping of the native promoter DNA sequence at theoriginal locus, as an additional copy of the gene under newtranscriptional regulation introduced as a genetic integration at aseparate locus, or as an additional copy on an episomal vector such as a2μ or centromeric plasmid.

1.6) The biosynthetic pathway in the engineered strain is improved bythe incorporation of a deletion or inactivating mutation of the nativeyeast gene ZWF1, which encodes an enzyme with glucose-6-phosphatedehydrogenase activity.

1.7) The biosynthetic pathway in the engineered strain is improved bythe incorporation of one or more deletion(s) or inactivating mutation(s)of known native alcohol dehydrogenase enzymes, such as the enzymesencoded by the genes ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, or SFA1.

1.8) The biosynthetic pathway in the engineered strain is improved bythe incorporation of one or more deletion(s) or inactivating mutation(s)of known native aldehyde oxidoreductases, such as ALD2, ALD3, ALD4.ALD5, or ALD6.

1.9) The biosynthetic pathway incorporates a heterologous enzyme for theconversion of tyrosine to L-DOPA. This enzyme may be from one of severalclasses, including, but not limited to bacterial tyrosinases, eukaryotictyrosinases, and tyrosine hydroxylases (Table 1). The gene for thisenzyme is incorporated as a genetic integration or on an episomal vectorsuch as a 2μ or centromeric plasmid. This L-DOPA producing enzyme isintroduced with engineered constitutive or dynamic regulation of proteinexpression by placing it under the control of a synthetic promoter.

1.9.1) In a biosynthetic pathway using a tyrosine hydroxylase enzyme forthe conversion of tyrosine to L-DOPA, additional expression of genesencoding enzymes for the synthesis and recycling of the pterin cofactortetrahydrobiopterin (BH4) and its derivatives are incorporated into theengineered strain in support of the activity of the tyrosine hydroxylaseenzyme. These enzymes include GTP cyclohydrolase,6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase,4a-hydroxytetrahydrobiopterin dehydratase, and quinoid dihydropteridinereductase (Table 1). The genes for these enzymes are incorporated as agenetic integration or on an episomal vector such as a 2μ or centromericplasmid. These BH4 synthesis and recycling enzymes are introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter.

1.10) The biosynthetic pathway incorporates a heterologous enzyme forthe decarboxylation of L-DOPA to produce dopamine. Enzymes with thisactivity are encoded by a genes from a variety of organisms includingbacteria, plants, and mammals. Examples include Pseudomonas putida DOPAdecarboxylase (PpDODC), Rattus norvegicus DOPA decarboxylase (RnDODC),and Papaver somniferum tyrosine/DOPA decarboxylase (PsTYDC) (Table 1).The gene for this enzyme is incorporated as a genetic integration or onan episomal vector such as a 2μ or centromeric plasmid. This dopamineproducing enzyme is introduced with engineered constitutive or dynamicregulation of protein expression by placing it under the control of asynthetic promoter.

1.11) A biosynthetic pathway for the production of 3,4-DHPA incorporatesa heterologous enzyme for the oxidation of dopamine to 3,4-DHPA.Examples of genes encoding this enzyme that may be used in the straininclude human monoamine oxidase A (hMAOA). E. coli monoamine oxidase(EcMAO), and Micrococcus luteus monoamine oxidase (MIMAO) (Table 1). Thegene for this enzyme is incorporated as a genetic integration or on anepisomal vector such as a 2μ or centromeric plasmid. This 3,4-DHPAproducing enzyme is introduced with engineered constitutive or dynamicregulation of protein expression by placing it under the control of asynthetic promoter.

1.11.1) Strains for the production of NC require dopamine and 4-HPA,while strains for the production of NL require dopamine and 3,4-DHPA,but not 4-HPA. A specific modification for the conversion of an NCproducing strain into an NL producing strain is the integration of a MAOgene into the yeast genome at the locus encoding the native yeast geneARO10. This combines a deletion of the native yeast enzyme responsiblefor converting a tyrosine biosynthetic precursor to 4-HPA with theintroduction of the enzyme capable of converting dopamine to 3,4-DHPA.

1.11.2) Yeast strains are constructed with a gene that expresses a MAOenzyme (1.11) under the control of an inducible promoter. When thestrain is grown in the presence of the inducer it can catalyze theconversion of dopamine to 3,4-DHPA, in the absence of inducer the strainonly produces 4-HPA.

1.11.2.1) Yeast strains are constructed with inducible MAO expression(1.11.2), where the inducible system also contains a DNA binding proteintargeted to the promoter regulating the ARO10 gene (1.3). The syntheticpromoter controlling ARO10 is therefore repressed when the promotercontrolling the MAO gene is activated and ARO10 is only expressed whenthe MAO gene is not transcriptionally active. This system allows for theconstruction of a single strain that conditionally only produces theprecursors for NC or NL.

B. NC-Producing Yeast Strains

Methods are developed to produce the BIA molecule NC in yeast anddemonstrate a first system for microbial synthesis of NC. With theengineered strains described herein, NC is produced and accumulated forits own value or combined with a biosynthetic pathway of additionalheterologous enzymes for the complete synthesis of downstream BIAs.

Specific Description:

2.1) Yeast strains are grown in liquid culture to a high cellconcentration before back diluting to intermediate concentrations (asmeasured by optical density or OD) in defined media containing highconcentrations of dopamine. The media components only need to satisfyconditions for growth of the strains; various growth feedstocks are used(for example, different sugars, nitrogen sources). The NC produced bythese yeast strains is excreted by the yeast cells and is measurable inthe spent media. Additional NC retained by cells is recovered via celllysis and extraction from the lysate.

2.2) Yeast strains containing various combinations of the modificationsas described in (1.1-1.8) substantially improve NC production from thatmeasurable in unmodified strains in fed dopamine assays as describedabove (2.1). In conditions where no extracellular tyrosine is availablein the yeast media, modifications described (1.1-1.8) provide forproduction of NC from fed dopamine; under these conditions the NCproduction from unmodified yeast strains is most often undetectable.

2.3) Yeast strains that produce NC when containing the modification asdescribed in (1.10) and grown as described in (2.1) when the additionalBIA precursor added to media is L-DOPA instead of dopamine.

2.3.1) Yeast strains as described in (2.3) containing variouscombinations of the modifications as described in (1.1-1.8)substantially improve production of NC.

2.4) Yeast strains that produce NC when containing both the heterologousenzymes for conversion of tyrosine to dopamine (1.9, 1.10) alongsidevarious combinations of modifications described above (1.1-1.8) andgrown in media without supplementation of tyrosine, L-DOPA, or dopamine.This specific example constitutes complete synthesis of NC by the strainfrom simple carbon and nitrogen sources.

2.5) Yeast strains are modified and cultured as described above(2.1-2.4) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme NCS, or truncated versions of the NCS enzyme,for the stereospecific catalysis of the reaction condensing dopamine and4-HPA for S-NC production. This enzyme may originate from one of severalplants, such as Papaver somniferum, Coptis japonica, and Thalicitumflavum (Table 1). The gene for this enzyme is incorporated as a geneticintegration or on an episomal vector such as a 2μ or centromericplasmid. This S-NC producing enzyme is introduced with engineeredconstitutive or dynamic regulation of protein expression by placing itunder the control of a synthetic promoter. The NC ultimately producedwill be an enantiomeric mixture with bias towards the S-stereoisomer.

C. NL-Producing Yeast Strains

Methods are developed to produce the BIA molecule NL from yeast. Withthe engineered strains described herein, NL is produced and accumulatedfor its own value or combined with a biosynthetic pathway of furtherheterologous enzymes for the complete synthesis of downstream BIAs.

Specific Description:

3.1) Yeast strains containing modifications as described in (1.11,1.11.1-1.11.2, 1.11.2.1) are grown in liquid culture as described in(2.1) produce NL.

3.2) Yeast strains containing various combinations of gene deletions asdescribed in (1.7.1.8) improve NL production from that measurable inunmodified strains in fed dopamine assays as described above (3.1).

3.3) Yeast strains that produce NL when containing the modifications asdescribed in (1.10, 1.11, 1.11.1-1.11.2, 1.11.2.1) and grown asdescribed in (3.1) when the additional BIA precursor is added to mediais L-DOPA instead of dopamine.

3.3.1) Yeast strains as described in (3.3) containing combinations ofgene deletions described in (1.7,1.8) improve production of NL.

3.4) Yeast strains that produce NL when containing both the heterologousenzymes for conversion of tyrosine to dopamine (1.9, 1.10) and dopamineto 3,4-HPA (1.11) alongside various combinations of modificationsdescribed above (1.1-1.8, 1.11.1) are grown in media withoutsupplementation of tyrosine, L-DOPA, or dopamine. This specific exampleconstitutes complete synthesis of NL by the strain from simple carbonand nitrogen sources.

3.5) Yeast strains modified and cultured as described above (3.1-3.4)where the biosynthetic pathway includes the incorporation of theheterologous enzyme NCS, or truncated versions of the NCS enzyme (Table1), for the stereospecific catalysis of the reaction condensing dopamineand 3,4-HPA for S-NL production. This enzyme may originate from one ofseveral plants, such as Papaver somniferum, Coptis japonica, andThalicitum flavum (Table 1). The gene for this enzyme is incorporated asa genetic integration or on an episomal vector such as a 2μ orcentromeric plasmid. This S-NL producing enzyme is introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter. The NC ultimatelyproduced is an enantiomeric mixture with bias towards theS-stereoisomer.

FIG. 1: Biosynthesis of Tyrosine and BIA Precursor Molecules

Schematic showing the biosynthetic pathway from glucose to tyrosine andother BIA precursors. Aromatic amino acid intermediates present innative yeast metabolism are written in black. Endogenous yeast enzymesare written in grey (apart from TYR, TyrH, DODC and MAO). Heterologousenzymes and non-natural BIA precursor molecules include TYR, TyrH. DODCand MAO. As described in (1.1, 1.2) wild-type yeast enzymes encoded byARO4 and ARO7 are allosterically inhibited by tyrosine, indicated hereby the dotted grey line. Individual steps in the pentose phosphosphatepathway and glycolysis are not explicitly detailed in this figure,although the genes TKL1 and ZWF1 (targeted in 1.5, 1.6) are involved inthe pentose phosphate pathway, as indicated.

FIG. 2: Effect of ZWF1 Knockout and TKL1 Over-Expression on PentosePhosphate Pathway (PPP)

Schematic detailing how modifications to TKL1 (1.5) and ZWF1 (1.6)affect the overall carbon flow through the pentose phosphate pathway inyeast when glucose is the primary carbon source. Panel A representswild-type carbon flow; Panel B represents the relative change in carbonflow in a modified strain.

FIG. 3A: Synthesis of NC from Precursor Molecules

NC is synthesized from one molecule of dopamine and one molecule of4-HPA via a Pictet-Spengler condensation reaction. This reaction canoccur spontaneously to produce a racemic mixture of R- and S-NC. Thisreaction can alternatively be catalyzed by the plant enzyme NCS, whichproduces S-NC.

FIG. 3B: Synthesis of NL from Precursor Molecules

NL is synthesized from one molecule of dopamine and one molecule of3,4-DHPA via a Pictet-Spengler condensation reaction. This reaction canoccur spontaneously to produce a racemic mixture of R- and S-NL. Whilethe natural product of NCS is NC, the enzyme has been shown to catalyzethe stereospecific production of S-NL (see e.g., Rueffer et al. (1981)(S)-Norlaudanosoline Synthase—the 1st Enzyme in the BenzylisoquinolineBiosynthetic-Pathway. Febs Lett 129(1):5-9).

Measurement of the BIA molecules is performed by LC-MS analysis, whereNC production (m/z=+272, 19.2 min retention time) and NL production(m/z=+288, 18.9 min retention time) were observed, with ion MS2fragmentation agreeing with both standards and published detectionmethods (see e.g., Schmidt et al. (2007) Poppy alkaloid profiling byelectrospray tandem mass spectrometry and electrospray FT-ICR massspectrometry after [ring-13C6]-tyramine feeding. Phytochemistry68(2):189-202).

FIG. 4: Effect of Four Genetic Modifications on NC Production withVarying Fed Tyrosine

NC production was demonstrated at several concentrations of fedtyrosine, including no fed tyrosine, in strains with targeted geneticmodifications. Wild-type strain, CEN.PK2, was integrated with constructsconferring one of four genetic changes (as described in 1.1-1.4):overexpression of ARO10 by promoter replacement with P_(TEF1),overexpression of ARO9 by promoter replacement with P_(TEF1),chromosomal integration of an ARO4^(FBR) allele, and chromosomalintegration of an ARO7^(FBR) allele. When incorporated alone, only theP_(TEF1)-ARO10 and ARO4^(FBR) increase production of NC. While boththese modifications increased NC production at all tyrosineconcentrations, the ARO^(FBR) integrated strain improved mostdrastically at zero fed tyrosine.

FIG. 5: NC Production with Combinations of Genetic Modifications

Some genetic modifications as described above (1.5, 1.6) improve NCproduction only in combination with the integration of the ARO4^(FBR)mutant (1.1). This figure shows four strains engineered with singlegenetic modifications, P_(TEF1)-ARO10 (1.3), P_(GPO)-TKL1 (1.5). ZWF1knockout (1.6), and ARO4^(FBR) (1.1), alongside three strainsconstructed with combinations of genetic modifications; Strain A(P_(GPD)-TKL1, ARO4^(FBR)), Strain B (ZWF1 knockout, ARO4^(FBR)), andStrain C (P_(GPD)-TKL1, ZWF1 knockout, ARO4^(FBR)). NC production isshown normalized to the WT strain, with Strain C exhibiting a five-foldincrease in NC production.

FIG. 6: NL Production in ALD/ADH Knockout Strains

NL production is improved by the deletion of competing yeast enzymes(1.7,1.8) in a strain expressing human MAOA on a 2μ plasmid (1.11) andgrown in media containing dopamine. NL production is shown as titermeasured in spent media normalized to the WT (with hMAO, but with nodeletions). Improvements in production is as much as ten times WT NLproduction.

FIG. 7: Activity of a DODC Enzyme In Vivo

Yeast strains transformed with DNA to express Papaver somniferumtyrosine/DOPA decarboxylase can convert L-DOPA to dopamine in vivo.Strains harboring a 2μ plasmid were grown in selective media and thenback-diluted into media containing L-DOPA. Spent media was then measuredfor concentrations of L-DOPA (retention time 4.8 min, m/z=+198) anddopamine (retention time 4.2 min, m/z=+154).

FIG. 8

NC is produced as described in (2.1) in multiple wild-type yeast labstrains at varying tyrosine concentrations. Specifically, each yeaststrain is inoculated into separate liquid cultures and grown overnightto OD₆₀₀˜10, then back-diluted in YNB minimal media without tyrosine toan OD₆₀₀˜1 and grown for 3 hours, 100 μl of each culture was mixed into400 μl YNB media containing 100 mM dopamine and varying concentrationsof tyrosine; each strain was grown in each media condition in triplicatesamples. NC titer was measured from culture supernatant on an LC-MSinstrument detecting m/z+272 ion count in peaks as described by (seee.g., Schmidt et al. (2007) Poppy alkaloid profiling by electrospraytandem mass spectrometry and electrospray FT-ICR mass spectrometry after[ring-13C6]-tyramine feeding. Phytochemistry 68(2):189-202). The area ofeach peak was integrated to calculate a relative quantity of the NC ineach sample and the results were normalized to the ion count area inCEN.PK2 yeast culture with 0 mg/L tyrosine.

FIG. 9

NC is produced as described In (2.1) in multiple engineered yeaststrains at fed 100 mM dopamine and no tyrosine. These data weregenerated in a separate experiment from those in FIG. 5. The strainsCSY977-981 were engineered to contain combinations of the geneticmodifications described in (1.1-1.6); the labels underneath each strainname indicate which modifications were incorporated into each strain.Strain CSY981 contains five genetic modifications to yeast nativemetabolism and exhibits a twelve-fold increase in NC titer above thewild-type yeast strain CEN.PK2.

FIG. 10

NC production as described in (2.1) for black diamonds and as describedin (2.3) for gray circles. Here NC was produced in an engineered yeaststrain (CSY980) with the additional integration of the L-DOPAdecarboxylase PpDODC (1.10). In separate liquid cultures this yeaststrain was grown in YNB minimal media containing varying concentrationsof dopamine (black diamonds) and YNB minimal media containing L-DOPA(gray circles; cultures not fed dopamine). The solid black linerepresents a linear regression of the relationship between the measuredNC and fed dopamine. The peak area measurements for the L-DOPA fedsamples were plotted along the regression line for dopamine fed samplesto show an “equivalent fed dopamine” quantity for the cultures fedL-DOPA. L-DOPA media was mixed to achieve a target concentration of 10mM L-DOPA, however L-DOPA was not fully soluble at that concentration,and the effective concentration of dissolved L-DOPA is estimated to beapproximately 6 mM. Based on the average NC titers of the two L-DOPA fedyeast cultures the “equivalent fed dopamine” concentration isapproximately 50 mM or 8× the fed L-DOPA concentration (indicated by thegray dotted lines).

FIG. 11

Mammalian tyrosine hydroxylases (TyrHs) are capable of hydroxylatingtyrosine, but are dependent on the co-substrate tetrahydrobiopterin(BH4) for activity, as described in (1.9.1). During the catalysis oftyrosine to L-DOPA by TyrH, molecular oxygen is split and transferred totyrosine and BH4, as shown by reaction 1. BH4 is oxidized toBH4-4α-carbinolamine (4αOH-8H4). Two heterologous enzymes are expressedin yeast to synthesize BH4 from the folate synthesis pathwayintermediate, dihydroneopterin triphosphate. First,6-pyruvoyltetrahydropterin synthase (PTPS) converts dihydroneopterin toPTP (reaction 2), which is then reduced to 8H4 by sepiapterin reductase(SepR, reaction 3). Two enzymes are responsible for the regeneration ofBH4 from its 4α-carbinolamine form. First, pterin-4a-carbinolaminedehydratase (PCD) catalyzes a loss of water reaction to formdihydrobioterin (reaction 4). Dihydrobiopterin is then reduced totetrahydrobiopterin by quinoid dihydropteridine reductase (QDHPR,reaction 5).

FIG. 12

Tyrosine hydroxylases expressed from yeast cells convert tyrosine toL-DOPA. Yeast strains transformed with plasmids carrying tyrosinehydroxylases from human (hTH2) and rat (RnTyrH) were grown in liquidmedia and then lysed in buffer containing tyrosine and the co-substrateBH4. After 6-hour incubations at 30° C., L-DOPA was measured in thelysate mixture by LC-MS. (A) LC-MS chromatogram confirms conversion oftyrosine to L-DOPA dependent on the presence of the co-substrate, BH4.(B) Fragmentation of the +198 m/z ion peak further confirms the presenceof L-DOPA in lysate samples. (See e.g., Lv et al. (2010) LC-MS-MSSimultaneous Determination of L-Dopa and its prodrug n-PentylHydrochloride in Rat Plasma. Chromatographia, 72(3/4), 239-243).

FIG. 13

Co-expression of tyrosine hydroxylase with a 8H4 biosynthetic enzymeenables conversion of tyrosine to L-DOPA in yeast cell lysatesEngineered yeast strains integrated with constructs expressing rattyrosine hydroxylase (RnTyrH) and rat sepiapterin reductase (RnSepR)were grown in liquid media and then lysed in buffer containing tyrosine,NADPH, and the BH4 biosynthetic precursor, sepiapterin. Co-expression ofa TyrH with the BH4 biosynthesis gene provides for activity of thetyrosine hydroxylase in the absence of BH4, but in the presence of theBH4 precursor, sepiapterin.

FIG. 14

Synthesis of the BIA molecules coclaurine and N-methylcoclaurine fromNC. Conversion of NC to downstream BIA molecules by plantmethyltransferase enzymes extends microbial BIA synthesis. Theseparticular BIA molecules can only be synthesized via a NC-dependentbiosynthesis scheme.

FIG. 15

Engineered yeast strains produce NC-derived BIA molecules from L-DOPA inliquid culture. A copy of PpDODC was integrated into the engineeredyeast strain, CSY979 (as described in FIG. 9), providing for theproduction of NC from L-DOPA (panel A, bottom chromatogram). Next a copyof the opium poppy 6-O-methyltransferase (Ps6OMT) gene was integratedinto this yeast strain to enable the production of coclaurine fromL-DOPA (panel A, middle chromatogram). Finally, a copy of both Ps60OMTand the opium poppy coclaurine-N-methyltransferase (PsCNMT) genes wereintegrated into the CSY979 yeast strain carrying the PpDODC gene toenable the production of N-methylcoclaurine from L-DOPA (panel A, topchromatogram). Both the NC and coclaurine measurements matchedchromatograms from chemical standards. The production ofN-methylcoclaurine was further confirmed by matching the fragmentationpattern of the +300 m/z ion peak to patterns in published literature(Panel B). (see e.g., Schmidt at al. (2007) Poppy alkaloid profiling byelectrospray tandem mass spectrometry and electrospray FT-ICR massspectrometry after [ring-13C6]-tyramine feeding. Phytochemistry68(2):189-202).

TABLE 1 Genes of interest as components of the engineered metabolicpathways Coding Specific Source Engineered sequence description EnzymeAbbrev. Catalyzed reactions organisms regulaton changes Genbank # ref.3-deoxy-d-arabinose- ARO4, erythrose-4-phosphate + Saccharomyces native,Feedback CAA85212.1 1.1 heptulosonate-7- DHAP PEP → DHAP (EC cerevisiaeconstitutive, inhibition phosphate synthase synthase 2.5.1.54) syntheticresistant regulation mutation, K229L, Q166K Chorismate mutase ARO7chorismate → Saccharomyces native, Feedback NP_015385.1 1.2 prephenatecerevisiae constitutive, inhibition (EC 5.4.9.5) synthetic resistantregulation mutation, T226I Phenylpyruvate ARO10 hydroxyphenyl-Saccharomyces constitutive NP_010668.3 1.3 decarboxylase pyruvate →cerevisiae overexpression, 4HPA (EC 4.1.1.80) synthetic regulationAromatic ARO9 hydroxyphenyl- Saccharomyces constitutive AEC14313.1 1.4aminotransferase pyruvate + cerevisiae overexpression, glutamate →synthetic tyrosine + regulation alpha-ketogluterate (EC 2.6.1.57)Transketolase TKL1 fructose-6-phosphate + Saccharomyces constitutiveNP_015399.1 1.5 glyceraldehyde-3- cerevisiae overexpression, phosphate ↔xylulose-5- synthetic phosphate + erythrose-4- regulation phosphate (EC2.2.1.1) Glucose-6- ZWF1 glucose-6-phosphate → Saccharomyces fullCAA96146.1 1.6 phosphate 6-phosphogluconolactone cerevisiae deletiondehydrogenase (EC 1.1.1.49) of coding region Alcohol ADH2-7 3HPA →tyrosol (EC Saccharomyces full NP_014032.1, 1.7 dehydrogenase SFA11.1.1.90) cerevisiae deletion AAT93007.1, of NP_011258.2, codingNP_009703.3, region NP_014051.3, NP_010030.1, NP_01013.1 4HPA →Saccharomyces full NP_013893.1, 1.8 Aldehyde oxidase ALD2-6hydroxyphenylacetic acid cerevisiae deletion NP_013892.1, (EC 1.2.1.39)of NP_015019.1, coding NP_010996.2, region NP_015264.1 Tyrosinase TYRtyrosine → L-DOPA, L- Ralstonia constitutive NP_518458.1, 1.9 DOPA →dopaquinone solanacearum, overexpression, AJ223816, (EC 1.14.18.1)Agaricus synthetic bisporus regulation Tyrosinase TyrH tyrosine → L-DOPAHomo constitutive NM 012740, 1.9 hydroxylase (EC 1.14.16.2) sapiens,overexpression, NM 000240, norvegicus synthetic Mus musculus regulationGTP cyclohydrolase FOL2 GTP → dihydroneopterin Saccharomyces nativeCAA97297.1, 1.9.1 triphosphate (EC 3.5.4.16) cerevisiae, regulation,NP_001019195.1, Homo constitutive NP_032128.1 sapiens, overexpression,Mus musculus synthetic regulation 6-pyruvoyl PTPS dihydroneopterinRattus constitutive AAH59140.1, 1.9.1 tetrahydrobiopterin triphosphate →PTP norvegicus, overexpression, BAA04224.1, (PTP) (EC 4.2.3.12) Homosynthetic AAH29013.1 synthase sapiens, regulation Mus musculusSepiapterin SepR PTP → BH4 Rattus constitutive NP_062054.1, 1.9.1reductase (EC 1.1.1.153) norvegicus, overexpression, NP_003115.1, Homosynthetic NP_035597.2 sapiens, regulation Mus musculus 4a- PCD 4a-Rattus constitutive NP_001007602.1, 1.9.1 hydroxytetra- hydroxytetra-norvegicus, overexpression, AAB25581.1, hydrobiopterin hydrobiopterin →Homo synthetic NP_079549.1 (pterin-4α- H2O + quinoid sapiens, regulationcarbinolamine) dihydropteridine Mus musculus dehydratase (EC 4.2.1.96)Quinoid QDHPR quinoid Rattus constitutive AAH72536.1, 1.9.1dihydropteridine dihydropteridine → norvegicus, overexpression,NP_000311.2, reductase BH4 (EC 1.5.1.34) Homo synthetic AAH02107.1sapiens, regulation Mus musculus L-DOPA DODC L-DOPA → dopaminePseudomonas constitutive AE015451.1, 1.10 decarboxylase (EC 4.1.1.28)putida, overexpression, NP_001257782.1 Rattus synthetic norvegicusregulation Tyrosine/DOPA TYDC L-DOPA → dopamine Papaver constitutive1.10 decarboxylase (EC 4.1.1.28) somniferum overexpression, syntheticregulation Monoamine MAO dopamine → 3,4-DHPA E. coli, constitutiveJ03792, D2367, 1.11, oxidase (EC 1.4.3.4) Homo overexpression,AB010716.1 1.11.1 sapiens, synthetic Micrococcus regulation luteusNorcoclaurine NCS 4HPA + dopamine → Coptis constitutive N-terminalBAF45337.1, 2.5, synthase S-norcoclaurine japonica, truncationACI45396.1, 3.5 (EC 4.2.1.78) Papaver ACO90258.1, somniferum,ACO90247.1, Papaver AEB71889.1 bracteatum, Thalicitum flavum, Corydalissaxicola Norcoclaurine 6-O- 6OMT Norcoclaurine → Papaver constitutiveAY268894 methyltransferase coclaurine somniferum, overexpression,AY610507 Norlaudanosoline → Thalicitum synthetic D298113′hydroxycoclaurine flavum, regulation ACO90225.1 EC 2.1.1.128 CoptisBAM37634.1 japonica, Papaver bracteatum, Eschscholzia californicaCoclaurine- CNMT Coclaurine → N- P. constitutive AY217336Nmethyltransferase methylcoclaurine somniferum, overexpression, AY6105083′hydroxycoclaurine → T. synthetic AB061863 3′-hydroxy- flavum,regulation Nmethylcoclaurine C. EC 2.1.1.140 japonica 4′-O- 4′OMT3′-hydroxy-N- P. constitutive AY217333, methyltransferasemethylcoclaurine → somniferum, overexpression, AY217334 Reticuline T.synthetic AY610510 EC 2.1.1.116 flavum, regulation D29812 CoptisBAM37633.1 japonica, Eschscholzia californica

Notwithstanding the appended clauses, the disclosure set forth herein isalso defined by the following clauses:

1. A host cell that produces a benzylisoquinoline alkaloid (BIA)precursor, wherein the host cell comprises one or more modificationsselected from the group consisting of:

one or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the cell;

one or more transcriptional modulation modifications of one or morebiosynthetic enzyme gene native to the cell;

one or more inactivating mutations in one or more enzymes native to thecell; and

one or more heterologous coding sequences that encode one or moreenzymes:

wherein when the cell comprises one or more heterologous codingsequences that encode one or more enzymes, it comprises at least oneadditional modification selected from the group consisting of: afeedback inhibition alleviating mutation in a biosynthetic enzyme genenative to the cell; a transcriptional modulation modification of abiosynthetic enzyme gene native to the cell; and an inactivatingmutation in an enzyme native to the cell.

2. The host cell according to Clause 1, wherein the BIA precursor isselected from the group consisting of norcoclaurine (NC) andnorlaudanosoline (NL).3. The host cell according to Clause 2, wherein the BIA precursor isnorcoclaurine (NC).4. The host cell according to Clause 2, wherein the BIA precursor isnorlaudanosoline (NL).5. The host cell according to Clause 1, wherein when the cell comprisesone or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the cell, it comprises a least oneadditional modification selected from the group consisting of: atranscriptional modulation modification of a biosynthetic enzyme genenative to the cell; an inactivating mutation in an enzyme native to thecell; and a heterologous coding sequence that encode an enzyme.6. The host cell according to Clause 1, wherein when the cell comprisesone or more transcriptional modulation modifications of one or morebiosynthetic enzyme genes native to the cell, it comprises at least oneadditional modification selected from the group consisting of: afeedback inhibition alleviating mutation in a biosynthetic enzyme genenative to the cell; an inactivating mutation in an enzyme native to thecell; and a heterologous coding sequence that encodes an enzyme.7. The host cell according to Clause 1, wherein when the cell comprisesone or more inactivating mutations in one or more enzymes native to thecell, it comprises at least one additional modification selected fromthe group consisting of: a feedback inhibition alleviating mutation in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and a heterologous coding sequence that encodes an enzyme.8. The host cell according to Clause 1, wherein the cell comprises oneor more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the cell.9. The host cell according to Clause 8, wherein the one or morebiosynthetic enzyme genes encode one or more enzymes selected from a3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and achorismate mutase.10. The host cell according to Clause 9, wherein the one or morefeedback inhibition alleviating mutations are present in a biosyntheticenzyme gene selected from ARO4 and ARO7.11. The host cell according to Clause 10, wherein the one or morefeedback inhibition alleviating mutations are present in the ARO4 gene.12. The host cell according to Clause 9, wherein the mutations areselected from mutation of position 229 and mutation of position 166 ofthe DAHP.13. The host cell according to any one of the preceding clauses, whereinthe cell overproduces one or more BIA precursor molecules.14. The host cell according to Clause 13, wherein the one or more BIAprecursor molecules are selected from the group consisting of tyrosine,4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.15. The host cell according to Clause 14, wherein the one or more BIAprecursor molecules are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) anddopamine.16. The host cell according to Clause 14, wherein the one or more BIAprecursor molecules are 4-hydroxyphenylacetaldehyde (4-HPA) anddopamine.17. The host cell according to Clause 1, wherein the cell comprises oneor more transcriptional modulation modifications of one or morebiosynthetic enzyme genes native to the cell.18. The host cell according to Clause 17, wherein the transcriptionalmodulation modification is substitution of a strong promoter for anative promoter of the one or more biosynthetic enzyme genes.19. The host cell according to Clause 18, wherein the one or morebiosynthetic enzyme genes is selected from the group consisting ofARO10, ARO9 and TKL.20. The host cell according to Clause 1, wherein the cell comprises oneor more inactivating mutations in one or more enzymes native to thecell.21. The host cell according to Clause 20, wherein the enzyme is aglucose-6-phosphate dehydrogenase.22. The host cell according to Clause 21, wherein the enzyme is ZWF1.23. The host cell according to Clause 20, wherein the cell comprises oneor more inactivating mutations in one or more enzymes native to the cellcomprising alcohol dehydrogenase activity.24. The host cell according to Clause 23, wherein the one or moreenzymes is selected from the group consisting of ADH2, ADH3. ADH4. ADH5.ADH6. ADH7 and SFA1.25. The host cell according to Clause 20, wherein the cell comprises oneor more inactivating mutations in one or more enzymes native to the cellcomprising aldehyde oxidoreductase activity.26. The host cell according to Clause 25, wherein the one or moreenzymes is selected from the group consisting of ALD2, ALD3, ALD4, ALD5,and ALD6.27. The host cell according to Clause 1, wherein the cell comprises oneor more heterologous coding sequences that encode one or more enzymes oractive fragments thereof.28. The host cell according to Clause 27, wherein the host cellcomprises an NC synthase activity.29. The host cell according to Clause 27, wherein the host cellcomprises a heterologous coding sequence for an NC synthase or activefragment thereof.30. The host cell according to Clause 29, wherein the one or moreenzymes or active fragments thereof convert tyrosine to L-DOPA.31. The host cell according to Clause 27, wherein the one or moreenzymes or active fragments thereof is selected from the groupconsisting of bacterial tyrosinases, eukaryotic tyrosineses and tyrosinehydroxylases.32. The host cell according to Clause 27, wherein the cell comprises oneor more heterologous coding sequences for one or more enzymes or activefragments thereof that convert L-DOPA to dopamine.33. The host cell according to Clause 27, wherein the cell comprises oneor more heterologous coding sequences for one or more enzymes or activefragments thereof that convert dopamine to 3,4-DHPA.34. The host cell according to Clause 33, wherein the one or moreenzymes is a monoamine oxidase (MAO).35. The host cell according to Clause 34, wherein the one or moreheterologous coding sequences comprises a MAO coding sequence integratedat a genomic locus encoding native ARO10.36. The host cell according to Clause 34, wherein the one or moreheterologous coding sequences comprises a MAO coding sequence operablylinked to an inducible promoter.37. The host cell according to Clause 368, wherein the induciblepromoter is part of an inducible system comprising a DNA binding proteintargeted to a promoter regulating the ARO10 gene.38. The host cell according to Clause 27, wherein the heterologouscoding sequence is from a source organism selected from the groupconsisting of P. somniferum, T. flavum and C. japonica.39. The host cell according to Clause 1, wherein the host cell is aeukaryotic cell.40. The host cell according to Clause 39, wherein the eukaryotic cell isa yeast cell.41. The host cell according to Clause 40, wherein the yeast cell is a S.cerevisiae cell, a Schizosaccharomyces pombe or a Pichia pastoris cell.42. The host cell according to Clause 41, wherein the yeast cell is a S.cerevisiae cell.43. A method of preparing a benzylisoquinoline alkaloid (BIA),comprising:

culturing a host cell according to any one of Clauses 1 to 42;

adding a growth feedstock to the cell culture; and

recovering the BIA from the cell culture.

44. The method according to Clause 43, wherein the BIA is a BIAprecursor selected from norlaudanosoline and norcoclaurine.45. The method according to Clause 43, further comprising adding astarting compound to the cell culture.46. The method according to Clause 43, further comprising producing aBIA precursor selected from reticuline, coclaurine. N-methylcoclaurineand norreticuline.47. The method according to Clause 43, further comprising producing oneor more opiate compounds.48. The method according to Clause 45, wherein the one or more opiatecompounds is selected from oripavine, morphine, codeine, hydromorphone,hydrocodone, oxycodone and oxymorphone from thebaine.49. A kit comprising:

a host cell according to any one of Clauses 1 to 42; and

one or more components selected from a starting compound, a growthfeedstock, a heterologous coding sequence, cloning vectors, multiplecloning sites (MCS), bi-directional promoters, an internal ribosomeentry site (IRES) and a cell culture media.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A host cell that produces a benzylisoquinolinealkaloid (BIA) precursor, wherein the host cell comprises one or moremodifications selected from the group consisting of: one or morefeedback inhibition alleviating mutations in one or more biosyntheticenzyme genes native to the cell; one or more transcriptional modulationmodifications of one or more biosynthetic enzyme gene native to thecell; one or more inactivating mutations in one or more enzymes nativeto the cell; and one or more heterologous coding sequences that encodeone or more enzymes; wherein when the cell comprises one or moreheterologous coding sequences that encode one or more enzymes, itcomprises at least one additional modification selected from the groupconsisting of: a feedback inhibition alleviating mutation in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and an inactivating mutation in an enzyme native to the cell. 2.The host cell according to claim 1, wherein the BIA precursor isselected from the group consisting of norcoclaurine (NC) andnorlaudanosoline (NL).
 3. The host cell according to claim 1, whereinthe cell comprises one or more feedback inhibition alleviating mutationsin one or more biosynthetic enzyme genes native to the cell.
 4. The hostcell according to any one of the preceding claims, wherein the celloverproduces one or more BIA precursor molecules.
 5. The host cellaccording to claim 4, wherein the one or more BIA precursor moleculesare selected from the group consisting of tyrosine,4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. 6.The host cell according to claim 1, wherein the cell comprises one ormore transcriptional modulation modifications of one or morebiosynthetic enzyme genes native to the cell.
 7. The host cell accordingto claim 6, wherein the transcriptional modulation modification issubstitution of a strong promoter for a native promoter of the one ormore biosynthetic enzyme genes.
 8. The host cell according to claim 1,wherein the cell comprises one or more inactivating mutations in one ormore enzymes native to the cell.
 9. The host cell according to claim 8,wherein the enzyme is a glucose-6-phosphate dehydrogenase, an alcoholdehydrogenase or an aldehyde oxidoreductase.
 10. The host cell accordingto claim 1, wherein the cell comprises one or more heterologous codingsequences that encode one or more enzymes or active fragments thereof.11. The host cell according to claim 1, wherein the host cell is aeukaryotic cell.
 12. The host cell according to claim 11, wherein theeukaryotic cell is a yeast cell.
 13. The host cell according to claim12, wherein the yeast cell is a S. cerevisiae cell, aSchizosaccharomyces pombe or a Pichia pastoris cell.
 14. A method ofpreparing a benzylisoquinoline alkaloid (BIA), comprising: culturing ahost cell according to any one of claims 1 to 13; adding a growthfeedstock to the cell culture; and recovering the BIA from the cellculture.